Mitochondria are required for a large number of cellular functions, including ion homeostasis, respiration, and programmed cell death. Consequently, defects in mitochondrial function have emerged as causative or contributing factors in a growing number of diverse human diseases such as cancer, cardiomyopathies, metabolic syndrome, and various neurodegenerative disorders. These diseases affect more than 50 million adults in the United States. Ad hoc proteomic and genetic studies in mammalian system have uncovered new mitochondrial protein (MP) complexes or pathways, but yet there has been no systematic experimental study of the mitochondrial interactome in mammalian system describing how these proteins function together in networks of pathways and complexes. Furthermore, it is also difficult to pinpoint the role of mitochondrial dysfunction in human disease because the interactions of MPs, both within and outside the mitochondria, are extensive and can be difficult to detect. This proposal seeks to begin addressing this deficit by creating a detailed physical (protein-protein) and genetic (gene-gene) interaction maps among MPs, which will help determine how mitochondrial protein complexes, within the framework of higher order networks, regulate and execute the associated processes. Using an optimized lentivirus-delivered tagging system coupled with mass spectrometry, a mammalian mitochondrial physical interactome map will be created using both native and mutant MPs with known association with human diseases. The resulting interaction networks will be compared to identify protein candidates that are relevant to disease onset and progression, and assess for variation in putative posttranslational modification sites involved in the progression of mitochondrial diseases (Aim1). Because genetic interaction (GI) is critical for revealing pathway-level relationships, optimized high precision quantitative pooled shRNAi coupled with deep sequencing approach, pioneered by the Weissman laboratory, will be used to query genes encoding for MP function by comparing the growth of pooled shRNAi treated cells on glucose relative to galactose as carbon sources in the media (Aim 2). This GI screening procedure will uncover new candidates that can toggle the glycolysis/mitochondrial respiration switch, which can be harnessed for therapeutic intervention. Finally, the GI data resulting from Aim 2 will be integrated with the proteomics data from Aim 1 (Aim 3) to investigate the functional relatedness and overall pathway architecture of the MP complexes to understand the fundamental mitochondrial biology and the role of mitochondrial dysfunction in disease. Collectively, these objectives are designed to provide new insights into the complex etiologies of diseases and have the potential to identify novel therapeutic avenues.